1. Field of the Invention
The invention relates to the chemical synthesis of oligonucleotides and to chemical entities useful in such synthesis. More specifically, the invention relates to sulfur transfer reagents capable of converting P(III) internucleosidic linkages of oligonucleotides to P(V) phosphorothioate linkages in solution or on solid phase.
2. Summary of the Related Art
Oligonucleotides and modified oligonucleotides are molecular tools of indispensable importance for research in molecular biology and for a variety of diagnostic and pharmaceutical applications including the use of siRNA and antisense inhibition of gene expression. Oligonucleotides that contain unnatural internucleoside linkages where one of the nonbridging oxygen atoms of the phosphate group is replaced by a sulfur atom are referred to as oligonucleotide phosphorothioates. Due to their enhanced nucleolytic stability, oligonucleotide phosphorothioates are among the most commonly used analogues. Their widespread use has led to an increasing demand for expeditious, inexpensive, and efficient methods for their preparation.
Synthesis of oligonucleotides is commonly performed on solid phase using well-established protocols employing phosphoramidite or H-phosphonate methods. Briefly, these approaches comprise anchoring the 3′-most nucleoside to a solid support functionalized with amino and/or hydroxyl moieties and subsequently adding the additional nucleotide residues in stepwise fashion. Internucleoside linkages are formed between the 3′ functional group of the incoming nucleoside and the 5′ hydroxyl group of the 5′-terminal nucleoside of the solid support-bound oligonucleotide. In the phosphoramidite approach, the internucleoside linkage is a protected phosphite moiety, whereas in the H-phosphonate approach, it is an H-phosphonate moiety. To convert these to the sulfur-containing phosphorothioate internucleoside linkage, the phosphite or H-phosphonate moieties are reacted with an appropriate sulfur transfer reagent. In the H-phosphonate approach, this sulfurization is carried out on all of the H-phosphonate linkages in a single step following the completion of oligonucleotide chain assembly, typically using elemental sulfur in a mixture of carbon disulfide and pyridine. In contrast, the phosphoramidite approach allows one to carry out a stepwise sulfurization following each coupling. Currently, the vast majority of oligonucleotides is synthesized using the phosphoramidite approach.
A number of sulfurization agents has been applied to the synthesis of oligonucleotide phosphorothioates. Examples of such agents include 3H-1,2-benzodithiol-3-one-1,1-dioxide, or Beaucage reagent (Iyer et al., J. Org. Chem. 1990, 55, 4693-4699), tetraethylthiuram disulfide (Vu et al., Tetrahedron Lett. 1991, 32, 3005-3008), dibenzoyl tetrasulfide (Rao et al., Tetrahedron Lett. 1992, 33, 4839-4842), bis(O,O-diisopropoxyphosphinothioyl) disulfide (Stec et al., Tetrahedron Lett. 1993, 33, 5317-5320), benzyltriethylammonium tetrathiomolybate (Rao et al., Tetrahedron Lett. 1994, 35, 6741-6744), bis(p-toluenesulfonyl) disulfide (Efimov et al., Nucl. Acids Res. 1995, 23, 4029-4033), 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) and 1,2,4-dithiazolidine-3,5-dione (U.S. Pat. No. 5,852,168), 3-amino-1,2,4-dithiazole-5-thione (U.S. Pat. No. 6,096,881), phenylacetyl disulfide (U.S. Pat. No. 6,242,591), 3-methyl-1,2,4-dithiazolin-5-one (Zhang et al., Tetrahedron Lett. 1999, 40, 2095-2098), 3-phenyl-1,2,4-dithiazoline-5-one (U.S. Pat. No. 6,500,944), and 3-amino-1,2,4-dithiazolidine-5-one (US 2004-559782 20040405).
A widely used Beaucage reagent and EDITH are somewhat difficult to synthesize; their hydrolytic stability is rather low. Agents such as tetraethylthiuram disulfide display slow reaction kinetics and thus are less convenient in high-throughput and large scale applications. To date, only Beaucage reagent and TETD are commercially available.